The abuse and irrational use of tetracyclines (TCs) in human medicine and animal husbandry has become a serious concern, affecting the ecological environment and human health. The aim of this study was to develop a sensitive and selective method using fully automatic solid-phase extraction coupled with ultra-performance liquid chromatography-tandem mass spectrometry for the determination of twelve TCs in water. Four isotope-labeled internal standards for TCs were used to correct matrix effects. Several parameters affecting extraction efficiency were systematically optimized, and the optimum experimental conditions found were 1.0 L water sample with 0.5 g/L Na2EDTA (pH 3.0) extracted and enriched by CNW HLB cartridge and eluted by 4 mL of acetone:methanol (v/v, 1:1). The enrichment factors were up to 798-1059 but only requiring about 60 min per six samples. Under the optimized conditions, the linearity of the method ranged from 0.2 to 100 μg/L for 12 TCs, the detection limits were as low as 0.01-0.15 ng/L, and the recoveries were in the range of 70%-118%, with relative standard deviations less than 15%. The developed method can be successfully utilized for the determination of 12 TCs in pure water, tap water, river water, and mariculture seawater. In summary, three and six TCs were detected in river water and mariculture seawater, respectively, with total concentrations of 0.074-0.520 ng/L (mean 0.248 ng/L) and 0.792-58.369 ng/L (12.629 ng/L), respectively. Tetracycline (TC) and oxytetracycline (OTC) were the dominant TCs in river water, while doxytetracycline (DXC) and OTC were dominant in mariculture seawater.

Mercury (Hg) emitted from East Asian has increased the risk of Hg in China Marginal Seas for decades. However, the speciation of Hg (especially the bioavailable Hg) in these regions remains unclear. To address this problem, we analyzed total Hg (THg) and methylmercury (MeHg) in the sediment and porewater of Yellow sea (YS) and East China Sea (ECS) and determined the speciation of Hg using both improved BCR sequential extraction and isotope dilution (ID) techniques. Nearshore areas of YS and ECS exhibited higher THg levels in sediments and porewater, suggesting the significant contribution of terrestrial inputs. The spatial distribution of MeHg showed similar trends with THg, but the sites with higher MeHg concentrations did not align with those of THg. The improved BCR sequential extraction method showed the residual fraction dominated Hg content (∼44 %) in both systems, with a minor bioavailable carbonate fraction (1 %). The Spearman correlation analysis indicates that Eh and pH are the two factors significantly affected Hg bioavailability in the sediment. The bioavailability of Hg (estimated by the BCR method) showed a significant positive correlation with MeHg levels in the sediment (R²=0.47, P < 0.05), suggesting that BCR can be used to estimate the potential of Hg methylation in the sediment. However, the extent of bioavailable Hg in BCR and ID method were 1.15 ± 0.38 % and 29.5 ± 14.8 %, respectively, implying that Hg bioavailability may be underestimated by BCR techniques compared to ID methods (T-test, P < 0.01).

In this study, we demonstrate the applicability of nitrogen microwave inductively coupled atmospheric pressure mass spectrometry (MICAP-MS) for Ca, Fe, and Se quantification in human serum using isotope dilution (ID) analysis. The matrix tolerance of MICAP-MS in Na matrix was investigated, revealing that high Na levels can suppress the signal intensity. This suppression is likely due to the plasma loading and the space charge effect. Moreover, 40Ca and 44Ca isotopic fractionation was noted at elevated Na concentration. Nine certified serum samples were analyzed using both external calibration and ID analysis. Overestimation of Cr, Zn, As, and Se was found in the results of external calibration, which might result from C-induced polyatomic interference and signal enhancement, respectively. Further investigations performed with methanol showed a similar enhancement effect for Zn, As, and Se, potentially supporting this assumption. The mass concentrations determined with ID analysis show metrological compatibility with the reference values, indicating that MICAP-MS combined with ID analysis can be a promising method for precise Ca, Fe, and Se determination. Moreover, this combination reduces the influence of matrix effects, broadening the applicability of MICAP-MS for samples with complex matrixes.

The aim was to determine ileal endogenous nitrogen losses (ENL) and true ileal N-digestibility (TD-N) under non-steady-state conditions of the 15N-isotope dilution technique (15N-IDT), using diets generating low and high ENL and compare results to those obtained under steady-state conditions. Twelve growing pigs (mean LW 22.4 kg) fitted with a post-valve T-caecum cannula were fed an enzyme-hydrolysed casein (EHC)-based diet or an EHC diet + 4% quebracho tannins (QT) and were labelled via continuous 15N-leucine i.v. infusion or twice daily oral 15N-leucine administration. Digesta were collected daily over three consecutive hours with blood plasma sampled on the four consecutive days after cessation of 15N-labelling. There was a significant effect of sampling day on the dilution factor. Endogenous N losses were significantly lower for the EHC than the EHC+QT diet (2.41 vs. 8.69 g/kg DMI), while no significant effect of sampling day was observed. The TD-N of the EHC+QT diet did not differ from the TD-N of the EHC diet (95.1 vs. 92.0%). A significant effect of sampling day was observed for TD-N with day 1 and 2, being higher than day 4. Non-steady-state conditions overestimated ENL by 25-28% as compared to 3 h collections in steady-state conditions, but the relative overestimation was similar for the EHC diet as for the EHC+QT diet. TD-N did not differ significantly compared to 12 h steady-state measurements, but comparison to 3 h steady-state measurements showed that non-steady-state conditions overestimated TD-N for the EHC+QT diet by 9%. However, on day 4 this overestimation disappeared. Using the 15N-IDT during non-steady-state conditions can provide valuable additional data on endogenous N losses and TD-N.

Isotope dilution ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) is commonly used for trace analysis of polyfluoroalkyl and perfluoroalkyl substances (PFAS) in difficult matrices. Commercial nontargeted analysis of major PFAS where relative concentrations are obtained cost effectively is rapidly emerging and is claimed to provide comparable results to that of absolute quantification using matrix matched calibration and isotope dilution UHPLC-MS/MS. However, this remains to be demonstrated on a large scale. We aimed to assess the performance of a targeted absolute quantification isotope dilution LC-MS/MS assay versus a commercial nontargeted relative quantification assay for detection of three major PFAS in human blood. We evaluated a population-based cohort of 503 individuals. Correlations were assessed using Spearman\'s rank correlation coefficients (rho). Precision and bias were assessed using Bland-Altman plots. For perfluorooctane sulfonic acid, the median concentrations were 5.10 ng/mL (interquartile range [IQR] 3.50-7.24 ng/mL), the two assays correlated with rho 0.83. For perfluorooctanoic acid, the median concentrations were 2.14 ng/mL (IQR 1.60-3.0 ng/mL), the two assays correlated with rho 0.92. For perfluorohexanesulfonate, the median concentrations were 5.5 ng/mL (IQR 2.50-11.61 ng/mL), the two assays correlated with rho 0.96. The Bland-Altman statistical test showed agreement of the mean difference for the majority of samples (97-98%) between the two assays. Absolute plasma concentrations of PFAS obtained using matrix matched calibration and isotope dilution UHPLC-MS/MS show agreement with relative plasma concentrations from a nontargeted commercial platform by Metabolon. We observed striking consistency between the two assays when examining the associations of the three PFAS with cholesterol, offering additional confidence in the validity of utilizing the nontargeted approach for correlations with various health phenotypes.

OBJECTIVE: The standardization of cystatin C (CysC) measurement has received increasing attention in recent years due to its importance in estimating glomerular filtration rate (GFR). Mass spectrometry-based assays have the potential to provide an accuracy base for CysC measurement. However, a precise, accurate and sustainable LC-MS/MS method for CysC is still lacking.
METHODS: The developed LC-MS/MS method quantified CysC by detecting signature peptide (T3) obtained from tryptic digestion. Stable isotope labeled T3 peptide (SIL-T3) was spiked to control matrix effects and errors caused by liquid handling. The protein denaturation, reduction and alkylation procedures were combined into a single step with incubation time of 1 h, and the digestion lasted for 3.5 h. In the method validation, digestion time-course, imprecision, accuracy, matrix effect, interference, limit of quantification (LOQ), carryover, linearity, and the comparability to two routine immunoassays were evaluated.
RESULTS: No significant matrix effect or interference was observed with the CysC measurement. The LOQ was 0.21 mg/L; the within-run and total imprecision were 1.33-2.05 % and 2.18-3.90 % for three serum pools (1.18-5.34 mg/L). The LC-MS/MS method was calibrated by ERM-DA471/IFCC and showed good correlation with two immunoassays traceable to ERM-DA471/IFCC. However, significant bias was observed for immunoassays against the LC-MS/MS method.
CONCLUSIONS: The developed LC-MS/MS method is robust and simpler and holds the promise to provide an accuracy base for routine immunoassays, which will promote the standardization of CysC measurement.

Current analytical methods for amino acid (AA) analysis in ruminant nutrition are time-consuming and expensive. This study aimed to develop a method for AA analysis that is faster, more efficient, rugged, and accessible. Four representative matrixes were selected for method development and validation: milk, tissue, feed, and soy flour standard reference material from National Institute of Standards and Technology. Acid and alkaline hydrolysis were used to analyze 18 AA. Separation of AA was performed using a Z-HILIC column in an 18-min run coupled to a triple quadrupole LC/MS system in positive and negative electrospray ionization for identification and quantitation. The method was evaluated for recovery, precision, calibration curve linearity, and limits of detection (LODs) and limits of quantitation (LOQs) and applied to other feed samples. Good quantitation results were achieved for all AA, with coefficients of determination (R2) over 0.995; LODs at 0.2-28.2 and LOQs at 0.7-94.1 ng/mL; intraday and interday precision <14.9% relative standard deviation; blank recovery between 75.6 and 116.2%; and sample recovery between 75.6 and 118.0%. Overall, AA concentrations were similar to literature values, and there was a tendency for higher N recovery as AA. In conclusion, an efficient and robust method was validated to routinely analyze AA for appropriate characterization in diet formulation for dairy cattle.

A 30 year old man was found with no signs of life in front of the house. The cyanide concentration in blood and urine was determined five years after the man\'s death. What is more, a stability study was conducted for 730 days in an authentic casework blood sample. Sample preparation procedure included precipitation with methanol:water mixture, solid phase extraction (SPE) and derivatization with the use of PFB-Br (pentafluorobenzyl bromide). The sample was analyzed using GC-QqQ-MS/MS (gas chromatopraphy coupled with tandem mass spectrometry) isotope dilution method. Separation was done using a SH-RXI-5MS column (30 m x 0.25 mm, 0.25 µm). Detection of PFB-CN and PFB-13CN was achieved using a triple-quadrupole mass spectrometer with an electron ionization (EI) ion source in multiple reaction monitoring (MRM) mode. After 5 years from the man\'s death, cyanide concentration was: 1900 ng/mL in blood and 500 ng/mL in urine. Stability study performed in an authentic blood sample 6 and 7 years after the man\'s death revealed cyanide concentrations of 1898.2 ng/mL and 1618.7 ng/mL, respectively. While spectrophotometric and colorimetric methods recorded both decrease and increase in cyanide concentration over time, newer chromatographic methods mainly indicate a decrease. The studies presented in this paper seem to confirm this trend. However, in order to interpretate the results of cyanide concentration in biological material reliably, more research is still necessary.

Although oceans play a key role in the global selenium (Se) cycle, there is currently very little quantitative information available on the distribution of Se concentrations and Se speciation in marine environments. In general, determining Se concentration and speciation in seawater is highly challenging due to very low Se levels ((sub)ng⋅L-1), whereas matrix elements interfering Se pre-concentration and detection are up to the g⋅L-1 levels. In this study, we established a sensitive method for the determination of the various Se chemical fractions present in natural seawater, i.e. selenite (SeIV), selenate (SeVI), organic Se-II + Se0 and total Se, using species-specific isotope dilution gas chromatography coupled to inductively coupled plasma mass spectrometry (ID-GC-ICP-MS). We compared different derivatization reagents and optimized specific pre-treatment protocols, including a microwave assisted oxidation protocol for the determination of total Se and organic Se-II + Se0 using H2O2. To increase sensitivity, we developed an online pre-concentration method based on large volume injection (LVI) using a programmed temperature vaporization (PTV) inlet. Eventually, the developed method achieved low absolute and methodological detection limits, i.e., respectively, 0.1-0.3 pg and 0.9-3.1 ng.L-1 for the different fractions. The accuracy of our method was of 2% for a certified reference material (CRM) diluted in artificial seawater while the precision was better than 4% for a freshwater CRM in artificial seawater matrix as well as two common seawater CRMs certified for trace elements excluding Se. As a proof-of-concept, we quantified the various Se fractions in a large number of natural water samples from the Baltic and North Seas, encompassing a wide range of salinity (7-35 psu), which shows that its detection limits are sufficient to determine total Se, SeIV, SeVI and organic Se-II + Se0 concentrations in brackish and marine systems.

Safrole oxide (SAFO), a metabolite of naturally occurring hepatocarcinogen safrole, is implicated in causing DNA adduct formation. Our previous study first detected the most abundant SAFO-induced DNA adduct, N7-(3-benzo[1,3] dioxol-5-yl-2-hydroxypropyl)guanine (N7γ-SAFO-G), in mouse urine using a well-developed isotope-dilution high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (ID-HPLC-ESI-MS/MS) method. This study further elucidated the genotoxic mode of action of SAFO in mice treated with SAFO 30, 60, 90, or 120 mg/kg for 28 days. The ID-HPLC-ESI-MS/MS method detected N7γ-SAFO-G with excellent sensitivity and specificity in mouse liver and urine of SAFO-treated mice. Our data provide the first direct evidence of SAFO-DNA adduct formation in rodent tissues. N7γ-SAFO-G levels in liver were significantly increased by SAFO 120 mg/kg compared with SAFO 30 mg/kg, suggesting rapid spontaneous or enzymatic depurination of N7γ-SAFO-G in tissue DNA. Urinary N7γ-SAFO-G exhibited a sublinear dose response. Moreover, the micronucleated peripheral reticulocyte frequencies increased dose-dependently and significantly correlated with N7γ-SAFO-G levels in liver (r = 0.8647; p < 0.0001) and urine (r = 0.846; p < 0.0001). Our study suggests that safrole-mediated genotoxicity may be caused partly by its metabolic activation to SAFO and that urinary N7γ-SAFO-G may serve as a chemically-specific cancer risk biomarker for safrole exposure.